Novel metarhizium genus microorganism and the method for controlling the soil pests using the same

ABSTRACT

The present invention relates to a novel fungus of  Metarhizium  genus and a method for controlling soil pests using the same, more particularly to a novel  Metarhizium anisopliae  HY-2 and a microbial insecticide comprising the same and a method for controlling soil pests using the same. The  Metarhizium anisopliae  HY-2 can effectively be used to control the soil pests such as  Scarabaeidae.

FIELD OF THE INVENTION

The present invention relates to a novel fungus of Metarhizium genus anda method for controlling soil pests using the same, more particularly toa novel Metarhizium anisopliae HY-2 and a microbial insecticidecomprising the same and a method for controlling soil pests using thesame.

BACKGROUND ART OF THE INVENTION

Pests cause a great loss to major agricultural products and varioususeful plants every year. To control the pests, 1,205 kg/km²/year ofagricultural chemicals (pesticides) have been used in Korea since mid1990s, which is 4.6 fold much as average amount (263 kg) used in OECDcountries and 48 times much as a minimum 25 kg used in New Zealand.Mostly organic pesticides and chemical insecticides have been used,resulting in the destruction of ecosystem, undergroundwater-contamination, increased toxicity remaining in agriculturalproducts and the appearance of pests having strong resistance. Countriesbrought about an agreement to reduce organic pesticide quantity usedevery year about 50% until 2004 at Rio environmental conference held in1992. In fact, 46% of conventional organic insecticides, especially 68%in vegetables and fruits growing fields, were substituted withalternative pesticides in the United States in 1997, in order to controlpests with less damage to environment.

A pest controlling method using insect pathogenic microorganism is anenvironmental protective control means that works selectively for targetpests only, prohibits the appearance of pests having resistance, isavailable lastingly and makes possible to produce agricultural productsin good quality without residual toxicity and without harming human,animals, plants and environment. Thus, many advanced countries in thefield of agriculture including US have been separated insect pathogenicmicroorganisms suitable for their own countries' condition,mass-produced and put to use thereof actually.

Though the environmental protective biotic pesticide takes just about 5%of total pesticide market, it grows as fast as more than 20% every yearowing to the recognition of importance of biotic pesticide. So, bioticpesticides are expected to take huge part of the market in years. Whendomestic market is open for alternative pesticides, it will be takenover by countries that have already developed biotic pesticides usinginsect pathogenic microorganisms since Korea has depended on onlyorganic pesticides. Then, Korea's foreign exchange holdings will bedecreased and export of domestic agricultural products produced bychemical pesticides will be obstructed. In order to cope with suchdifficult situation effectively, it is urgently required to developbiotic (microbial) pesticides using insect pathogenic microorganismssuitable for our agricultural environment.

As a kind of soil pests, Mimela splendems (gold bug) is a euryphagouspest harming plants, especially plants of Rossaceae, Salicaceae,Fagaceae, Betulaceae and Aceraceae. Some adults of soil pests harmleaves of crops or plants and larvae do roots of various crops andgrass. The gold bugs damage not only various broad-leaved trees inforest but general crops like corns or tomatoes, fruits like apples orapricots, flowers like chrysanthemums or roses, tea plants, many herbs,ginseng farms, etc (Lee et al, Korean Journal of Applied Entomology,1997, 36, 2, 156-165).

The larvae of gold bugs developing and living in golf courses directlyharm grass roots to death and indirectly provide themselves as feed forbirds, causing digging up the grass. Therefore, they have a bad effecton the preservation of grass quality (Chu et al, Korean Journal ofTurfgrass Science, 1998, 12, 3, 225-236; Lee et al, Korean Journal ofApplied Entomology, 1997, 36, 2, 156-165; Lee, PhD Thesis, 2000). Tocontrol such soil insects like gold bugs, fenitrothion emulsion,chlorpyrifos-methyl emulsion and ethoprophos granules have been used.But those chemical pesticides have effects on only just-hatched larvae(the first larva stage). Thus, catching the right time is essential forcontrolling those insects. By the way, those chemical pesticides weakenthe grass and cause overdose (Korea Patent Application #1999-15472).

Great efforts have been made to control soil pests like gold bugs,Encarcia formosa, Eretmocerus eremicus, Plutella xylostella, Spodopteralitura and Nilaparvata lugens. But using the conventional chemicalpesticides causes not only high expense but also such problems that thedestruction of ecosystem, the under water contamination, the residualtoxicity in agricultural products and the appearance of insects havingresistance.

Thus, in order to develop an environmental protective controllingmethod, the present inventors have searched insect pathogenicmicroorganisms that are good for controlling soil pests especiallyharming major agricultural products and confirmed that a fungus ofMetarhizium genus kills soil pests including the larvae of gold bugs.So, the present inventors have completed the present invention bypreparing a microbial insecticide comprising the fungus of Metarhiziumgenus for controlling soil pests.

SUMMARY OF THE INVENTION

The present invention relates to a novel fungus of Metarhizium genus anda method for controlling soil pests using the same, more particularly toa novel Metarhizium anisopliae HY-2 and a microbial insecticidecomprising the same and a method for controlling soil pests using thesame. The Metarhizium anisopliae HY-2 can effectively be used to controlthe soil pests such as Scarabaeidae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electron microphotograph showing the Metarhizium anisopliaeHY-2 of the present invention,

FIG. 2 is a diagram showing the manufacturing process of a microbialinsecticide hydrate comprising the Metarhizium anisopliae HY-2 of thepresent invention for controlling soil pests,

FIG. 3 is a diagram showing the manufacturing process of a microbialinsecticide granule comprising the Metarhizium anisopliae HY-2 of thepresent invention for controlling soil pests,

FIG. 4 is a graph showing the growth curve of the Metarhizium anisopliaeHY-2 of the present invention,

FIG. 5 is a graph showing the insecticidal effect of the Metarhiziumanisopliae HY-2 of the present invention measured by dipping method,

FIG. 6 is a set of photographs showing the larva of an Adoretustenuimaculatus killed by the infection with the Metarhizium anisopliaeHY-2 of the present invention,

-   -   A: a photograph of the larva just infected by Metarhizium        anisopliae HY-2,    -   B: a photograph of the larva three days after being infected by        Metarhizium anisopliae HY-2,    -   C: a photograph of the larva seven days after being infected by        Metarhizium anisopliae HY-2

FIG. 7 is a graph showing the insecticidal effect of the Metarhiziumanisopliae HY-2 of the present invention measured by contact toxicitytest.

DETAILED DESCRIPTION OF THE INVENTION

To achieve the above object, the present invention provides a novelfungus of Metarhizium genus that is useful for controlling soil pestsharming major crops.

The present invention also provides a medium for mass-production of afungus of Metarhizium genus.

The present invention further provides a microbial insecticidecomprising the above fungus of Metarhizium genus for controlling soilpests and a preparation method thereof.

Further features of the present invention will appear hereinafter.

The present invention provides a novel microorganism Metarhiziumanisopliae HY-2 having specific insectisidal effect on soil pests.

The present inventors separated a microorganism having insecticidaleffect on soil pests from dead insects infected with insect pathogenicmicroorganisms and soil samples where soil pests were inhabited. Themicroorganism of the present invention was identified as a kind ofMetarhizium anisopliae (see FIG. 1). The microorganism of the presentinvention was named as “Metarhizium anisopliae HY-2” and deposited atGene Bank of Korea Research Institute of Biosciene and Biotehnology onMar. 10, 1995 (Accession No: KCTC 0156BP).

The present invention also provides a medium for economicalmass-production of the above fungus of Metarhizium genus.

The medium of the present invention was prepared by mixing carbonsource, nitrogen source and inorganic elements with water. Autoclave ispreferred for the preparation of the media.

As for a carbon source, glucose, cornstarch, saccharose, molasses, wheatbran, rice bran, etc were preferably used and wheat bran or rice branwas more preferred. As for a nitrogen source, yeast extract, soybeanflour, corn-immersion, malt extract, peptone, etc were preferably usedand yeast extract was more preferred. As for an inorganic element,potassium chloride (KCl), magnesium sulfonate (MgSO₄), ferroic sulfate(FeSO₄), sodium nitrate (NaNO₃), dipotassium phosphate (K₂HPO₄), etcwere preferably used and sodium nitrate was more preferred.

The present invention further provides a microbial insecticidecomprising a fungus of Metarhizium genus for controlling soil pests anda preparation method thereof.

Microbial insecticides of the present invention containing a fungus ofMetarhizium genus for controlling soil pests can be prepared in the formof either wettable powders or tablets.

The wettable powders of the above microbial insecticides can be obtainedby pulverization after drying solid media inoculated with Metarhiziumanisopliae HY-2 and mixing surfactant and diluent with it.

Surfactants used for preparing wettable powders of the above microbialinsecticides can be selected from a group consisting of polycarboxylate, sodium lingo sulfate, sodium dialkyl sulfosuccinate, sodiumalkyl sulfonate, polyoxy ethylene alkyl phenyl ether, sodiumtripolyphosphate, polyoxyethylene alkyl aryl phosphoric ester,polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl arylpolymer,polyoxyalkylon alkyl phenyl ether, polyoxyethylene nonyl phenyl ether,sodium sulfonate, naphthalene formaldehyde, triton 100 and tween 80.Diluents can be selected from a group consisting of soybean flour, rice,wheat, yellow earth and diatomaceous earth.

The granules of the above microbial insecticides contain spores ofMetarhizium anisopliae HY-2, surfactants, nutrients and disintegrators,and can include diluents additionally.

The tablets of microbial insecticides of the present invention arepreferably prepared with the following weight ratios: Metarhiziumanisopliae HY-2 spores 10-60 weight parts, surfactants 2-16 weightparts, nutrients 5-20 weight parts, disintegrators 10-30 weight parts.Inactive carriers, preservatives, wetting agents, supply accelerants,attractants, encapsulating agents, binders, emulsifiers, dyes, UVprotectors, buffers, fluids, etc can be additionally added for thepreparation of the tablets of microbial insecticides of the presentinvention.

Surfactants used for preparing tablets of the above microbialinsecticides can be selected from a group consisting of polycarboxylate, sodium lingosulfate, sodium dialkyl sulfosuccinate, sodiumalkyl sulfonate, polyoxyethylene alkyl phenyl ether, sodiumtripolyphosphate, polyoxyethylene alkyl aryl phosphoric ester,polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl arylpolymer,polyoxyalkylon alkyl phenyl ether, polyoxyethylene nonyl phenyl ether,sodium sulfonate and naphthalene formaldehyde.

Nutrients can be selected from a group consisting of dextrin, glucoseand starch, disintegrators can be selected from a group consisting ofbentonite, talc, dialite, kaolin and sodium carbonate, and diluents canbe selected from a group consisting of lignin, lignin sulfate andeatomaceus ether.

The preparation method for wettable powders of microbial insecticides ofthe present invention comprises the following steps:

-   -   1) Pulverizing after drying solid media inoculated with        Metarhizium anisopliae HY-2; and    -   2) Adding surfactants and diluents to the pulverized powders of        the above step 1) and then pulverizing thereof again (see FIG.        2).

And, the preparation method for granules of microbial insecticides ofthe present invention comprises the following steps:

-   -   1) Pulverizing after drying solid media inoculated with        Metarhizium anisopliae HY-2;    -   2) Adding surfactants, adjuvant and diluents to the pulverized        powders of the above step 1) and then kneading thereof with        water; and    -   3) Granulating the dough prepared in the above step 2) and then        drying thereof (see FIG. 3).

The present inventors tested if the medium of the present invention wassuitable for mass-production of Metarhizium anisopliae HY-2. As aresult, 10⁸ cells were obtained in the second week of culture and eventhough early growth rate of bacteria depended on the amount of inoculum,the bacteria were kept growing well regardless of the amount of inoculumas culture continued, suggesting that the medium of the presentinvention was suitable for mass-production of the bacteria (see FIG. 4).

The present inventors also investigated the insecticidal effect ofMetarhizium anisopliae HY-2 of the present invention on gold bugs usingdipping method. As a result, spore-suspension solution of Metarhiziumanisopliae HY-2 was confirmed to have excellent insecticidal effect(56-64%) while a control group showed just 13-26% insecticidal effect(see FIG. 5). In addition, the larvae of Adoretus tenuimaculatusinfected with Metarhizium anisopliae HY-2 were detected after a while(see FIG. 6).

The present inventors further confirmed the insecticidal effect ofMetarhizium anisopliae HY-2 of the present invention on gold bugs usingcontact toxicity test. As a result, Metarhizium anisopliae HY-2 wasconfirmed to have excellent insecticidal effect (60-64%) while a controlgroup showed just 20-27% insecticidal effect (see FIG. 7).

Therefore, the Metarhizium anisopliae HY-2 of the present invention wasproved to have excellent insecticidal effect on soil pests.

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that. those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Separation and Identification of Microorganisms

In order to separate microorganisms that are useful for controlling soilpests, the present inventors collected dead insects infected with insectpathogenic microorganisms and at the same time, picked insects from soilsamples where soil pests were inhibiting as well. Particularly, thepresent inventors crushed soil pests dead by infection with insectpathogenic microorganisms and diluted with sterile water. Took 0.1 ml ofsuspension and smeared on microorganism test medium (Dermatophyte testmedium) as presented in Table 1. Suspended 0.1 g of soil samples with 5ml of sterile water and smeared on microorganism test medium as well.Culture thereof at 30° C. for 5 days and made a selection, which wasinoculated on new DTM medium and further cultured under the samecondition above. TABLE 1 Ingredient Amount (%) Glucose 2 Soytone 1Phenol Red 0.02 Cycloheximide 0.05 Chloheximide 0.01 Gentamycine 0.01Agar 1.5 Acetone (w/w) 2

As a result, the present inventors separated microorganism showedexcellent controlling effect on soil pests, and identified thereof. Themicroorganism was identified as a kind of Metarhizium anisopliae (FIG.1), named as “Metarhizium anisopliae HY-2” and deposited at Gene Bank ofKorea Research Institute of Biosciene and Biotehnology on March 10, 1995(Accession No: KCTC 0156BP).

Example 2 Preservation and Cultivation of the Separated Microorganism

For the pre-culture of Metarhizium anisopliae HY-2 of the presentinvention separated in the above Example 1 for further mass-culture andpreservation, CDAY medium prepared by adding yeast extract to Czapekmedium (Czapek-Dox broth, DIFCO) was used (Table 2). TABLE 2 IngredientAmount (%) Bacto saccharose 3 Sodium nitrate 0.3 Diphotassium phosphate0.1 Mangnessium sulfate 0.05 Potassium chloride 0.05 Ferrious sulfate0.001 Yeast extract 0.5

CDAY plate medium containing 2% agar was used for the preservation ofmicroorganisms, which was cultured at 26° C. for about 15 days untilspores were formed. Cut the medium on which hyphae of microorganismswere blooming by sterilized stick-like test spoon with making blocks.Set the test spoon straight, so that three dimension on medium weretouched by that. Untouched one side was taken and preserved in acontainer containing distilled water including 15% glycerol at −70° C.

CDAY medium was autoclaved at 121° C. for 20 minutes, after whichbacteria were inoculated thereto and shaking-cultured at 26° C. with 180rpm for 4-5 days.

Example 3 Preparation of Medium for Mass-Production

The present inventors prepared a commercial medium for mass-productionof a fungus of Metarhzium genus of the present invention. Particularly,mixed 3 l of water, 4 kg of wheat bran and 2 kg of rice bran together inan automatic mixer for 6 minutes. Put the mixture in a polypropylene bag(50×22×10 cm²) for autoclave to which 2 sheets of filter membrane (7×7cm², effective area diameter 3.5 cm) were attached for air-permeability.After sealing the bag, performed autoclave at 121° C. for 30 minutes,resulting in the preparation of a commercial culture medium formass-production.

Example 4 Preparation of Wettable Powders of Microbial Insecticide

The present inventors prepared wettable powders (WP) that can be easilyhydrated as being diluted with water in order to provide Metarhiziumanisopliae HY-2 raw-powders stably. Particularly, heated the solidmedium prepared in Example 3 that was inoculated with Metarhiziumanisopliae HY-2 in a dry oven at 70° C. for 2 hours. Upon finishingdrying, pulverized thereof. Add 10% surfactants and 40-60% diluents tothe above 30-50% raw-powders and mixed well. Pulverized the mixture witha grinder (Jet-O-mill, Aljet) and then tested hydrating capacity for thepreparation of wettable powders (FIG. 2).

In order to confirm the stability of the microbial insecticides forcontrolling soil pests prepared above, performed preservation test onthe wettable powders of the present invention. As a result, themicrobial insecticides showed about 45% recovery rate (4.19×10⁸spores/g).

Example 5 Preparation of Microbial Insecticide Granules

The present inventors prepared granules (GR) that could be used as theywere in order to provide Metarhizium anisopliae HY-2 raw-powders stably.Particularly, heated the solid medium inoculated with Metarhiziumanisopliae HY-2 in a dry oven at 70° C. for 2 hours. After finishingdrying, pulverized thereof. Add 3% surfactants, 2% adjuvants and 10-30%diluents to the above 30-50% raw-powders and then mixed well. Kneadedthe mixture with 35% water. Upon finishing kneading, granulated thereof(diameter: 1 mm, length: 1-5 mm) using Basket type extruder (Fuji powderCo) and then dried in a dry oven at 70° C. Removed dusts by sieving thedried materials with a 16-30 mesh sieve, resulting in the preparation ofmicrobial insecticide granules (FIG. 3).

In order to confirm the stability of the microbial insecticides forcontrolling soil pests prepared above, performed preservation test onthe granules of the present invention. As a result, the microbialinsecticides showed about 30% recovery rate (7.14×10⁶ spores/g).

<5-1> Preparation of Microbial Insecticide Granules 1

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 15% sodium tripolyphosphate (Hannongchemicals Inc.), 15% polycarboxylate (Hannong chemicals Inc.) and 20%talc (Table 3). TABLE 3 Components Effective Granules IngredientSurfactants Nutrients Disintegrators 1 Spores of 50%Sodiumtripolyphosphate 15%  Talc 20% (Example HY-2 Polycarboxylate 15% 5-1) 2 Spores of 50% Polycarboxylate 3% Glucose 10% Bentonite 20%(Example HY-2 Sodiumlignosulfonate 3% Calciumcarbonate 13% 5-2) Sodiumdialkyl 1% sulfosuccinate 3 Spores of 50% Sodium alkyl aryl 5% Starch10% Talc 30% (Example HY-2 sulfonate 5-3) Polyoxyethylene 1% alkylphenylether Sodium dialkyl 1% sulfosuccinate 4 Spores of 50%Polycarboxylate 3% Starch 10% Bentonite 20% (Example HY-2Polyoxyethylene 1% Talc 13% 5-4) alkyl phenyl etherCalciumlignosulfonate 3% 5 Spores of 50% Polyoxyethylene 5% Glucose 10%Bentonite 20% (Example HY-2 alkyl aryl Talc 12% 5-5) phosphoric esterPolyoxyethylene 3% alkyl aryl ether and polyoxyethylene alkyl arylpolymer 6 Spores of 50% Polyoxyethylene 1% Staech 10% Bentonite 20%(Example HY-2 alkyl phenyl Talc 16% 5-6) ether Sodiumtripolyphosphate 3%7 Spores of 50% Polycarboxylate 3% Starch 10% Bentonite 20% (ExampleHY-2 Polyoxyethylene 5% Kaolin 12% 5-7) alkyl aryl ether andpolyoxyethylene alkyl aryl polymer 8 Spores of 50% Polycarboxylate 3%Starch 10% Dialite 20% (Example HY-2 Sodium alkyl 5% Calciumcarbonate11% 5-8) aryl sulfonate Sodium dialkyl 1% sulfosuccinate 9 Spores of 50%Sodiumtripolyphosphate 3% Starch 10% Bentonite 10% (Example HY-2Polyoxyethylene 5% Dextran 10% Dialite 12% 5-9) alkyl aryl phosphoricester 10 Spores of 50% Polyoxyethylene 5% Dextran  5% Bentonite 20%(Example HY-2 alkyl aryl Talc 12% 5-10) polymer specialCalciumlignosulfonate 5% Polycarboxylate 3% 11 Spores of 50%Polyoxyalkylon 3% Dextran  5% Calciumcarbonate 28% (Example HY-2 alkylphenyl Staech 10% 5-11) ether Sodiumtripolyphosphate 3% Sodium dialkyl1% sulfosuccinate 12 Spores of 50% Sodiumlignosulfonate 3% Glucose 10%Kaolin 20% (Example HY-2 Polyoxyethylene 3% Talc 14% 5-12) alkyl arylether polymer 13 Spores of 50% Polyoxyethylene 3% Dextran  5% Kaolin 20%(Example HY-2 nonyl phenyl Dialite 19% 5-13) ether Sodium sulfonate 2%naphthalene formaldehyde Sodium dialkyl 1% sulfosuccinate<5-2> Preparation of Microbial Insecticide Granules 2

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 3% polycarboxylate, 1% sodiumlignosulfonate, 10% glucose, 20% bentonite and 13% calcium carbonate(Table 3).

<5-3> Preparation of Microbial Insecticide Granules 3

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 5% sodium alkyl aryl sulfonate, 1%polyoxyethylene alkyl phenyl ether, 10% starch, 30% talc and 3% dialite(Table 3).

<5-4> Preparation of Microbial Insecticide Granules 4

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 3% polycarboxylate, 1%polyoxyethylene alkyl phenyl ether, 3% calcium lignosulfonate, 10%starch, 20% bentonite and 13% talc (Table 3).

<5-5> Preparation of Microbial Insecticide Granules 5

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 5% polyoxyethylene aryl phosphoricester, 3% polyoxyethylene alkyl aryl ether and polyoxyethylene alkylaryl polymer, 10% glucose, 20% bentonite and 12% talc (Table 3).

<5-6> Preparation of Microbial Insecticide Granules 6

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 1% polyoxyethylene alkyl phenylether, 3% sodium tripolyphosphate, 10% starch, 20% bentonite and 16%talc (Table 3).

<5-7> Preparation of Microbial Insecticide Granules 7

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 3% polycarboxylate, 5%polyoxyethylene alkyl aryl ether and polyoxyethylene alkyl aryl polymer,10% starch, 20% bentonite and 12% kaolin (Table 3).

<5-8> Preparation of Microbial Insecticide Granules 8

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 3% polycarboxylate, 5% sodium alkylaryl sulfonate, 1% sodium dialkyl sulfosuccinate, 10% starch, 20%dialite and 11% calcium carbonate (Table 3).

<5-9> Preparation of Microbial Insecticide Granules 9

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 3% sodiumtripolyphosphate, 5%polyoxyethylene alkyl aryl phosphoric ester, 10% starch, 10% dextrin,10% bentonite and 12% dialite (Table 3).

<5-10> Preparation of Microbial Insecticide Granules 10

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 5% polyoxyethylene alkyl arylpolymer special, 5% calcium lignosulfonate, 3% polycarboxylate, 5%dextrin, 20% bentonite and 12% talc (Table 3).

<5-11> Preparation of Microbial Insecticide Granules 11

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 3% polyoxyalkylone alkyl phenylether, 3% sodium tripolyphosphate, 1% sodium dialkyl sulfosuccinate, 5%dextrin, 10% starch and 28% calcium carbonate (Table 3).

<5-12> Preparation of Microbial Insecticide Granules 12

The present inventors prepared granules having the same effect as themicrobial insecticide granules for controlling soil pests prepared inthe above S Example 5. Precisely, the present inventors preparedmicrobial insecticides granules for controlling soil pests consisting of50% Metarhizium anisopliae HY-2 spores, 3% sodium lignosulfonate, 3%polyoxyethylene alkyl aryl ether polymer, 10% glucose, 20% kaolin and11% talc (Table 3).

<5-13> Preparation of Microbial Insecticide Granules 13

The present inventors prepared granules having the same effect as themicrobial insecticide granules is for controlling soil pests prepared inthe above Example 5. Precisely, the present inventors prepared microbialinsecticides granules for controlling soil pests consisting of 50%Metarhizium anisopliae HY-2 spores, 3% polyoxyethylene nonyl phenylether, 2% sodium sulfonate naphthalene formaldehyde, 1% sodium dialkylsulfosuccinate, 5% dextran, 5% kaolin and 1% dialite (Table 3).

Example 6 Preparation of Formulations for Capsulation of MicrobialInsecticide

The present inventors prepared formulations for capsulation of microbialinsecticide having the same effect as the microbial insecticide granulesfor controlling soil pests prepared in the above Example 5.Particularly, mixed 11% soybean flour, 6% mung beans, 4% rice flour, 4%potato starch, 4% rye flour and 3% yellow earth together and autoclavedthereof at 121° C. for 20 minutes. Before completely cooling down, mixedthereof well again and then cool it down in cold water. Mixed thoroughlyover 1 minute using a stirrer (d=5 cm, over 200 rpm). While keeping onstirring, added additives and Metarhizium anisopliae HY-2 spores andmixed well again, resulting in the preparation of formulations forcapsulation of microbial insecticide for controlling soil pests.

Example 7 Security of Test Insect

The present inventors used Adoretus tenuinaculatus among gold bugs, akind of soil pests, as a test insect. The imagoes (adult insects) werecollected in Yusung country club, Taejeon, Korea and bred throughgenerations using a chestnut tree as a host plant. Tests were donewithin 2-4 generations. The larvae were bred with artificial feeds(sawdust:bed soil:water=5:3:2). Both imagoes and larvae of the testinsects were bred in cages (30 cm×30 cm×50 cm) in which temperature wasmaintained at 25±2° C. and relative humidity was kept 60-70%.

Experimental Example 1 Mass-Culture of a Fungus of Metarhizium Genus

The present inventors confirmed if the medium prepared in the aboveExample 3 was suitable for mass-production of Metarhizium anisopliaeHY-2 of the present invention. Particularly, after liquid culturingMetarhizium anisopliae HY-2 that was stored at −70° C. in wheat branmedium (wheat bran 333 g, rice bran 167 g, 1^(st) distilled water 250ml), inoculated thereof on the medium for mass-production prepared inExample 3 for further culture. Inoculated 2 ml (7.76×10³ cfu/g) and 10ml (3.38×10⁴ cfu/g) of pre-cultured solution onto the 750 g of mediumfor mass-production of the present invention respectively, and thencultured them in a culture room for 3-4 weeks in which the temperaturewas maintained at 27° C., the radiation intensity was kept 40 W×2×1.5and the relative humidity was kept 40-70%. Every 7 days afterinoculation, the number of live bacteria was counted, so that the growthof Metarhizium anisopliae HY-2 according to the amount of inoculum couldbe observed. Specific growth rate (μ max) was measured based on theincreased density of live bacteria. To calculate the specific growthrate, subtracted early density of live bacteria from late density oflive bacteria, which was converted by log value and then subtractedbeginning time from after-growth time therefrom, suggesting the growthrate of the number of live bacteria per hour.

As a result, when inoculated 2 ml of inoculum at the early stage, thegrowth speed for the early 2 weeks was 0.028 hr⁻¹ and when inoculated 10ml of inoculum, the growth speed for the early weeks was 0.034 hr⁻¹. Andthe present inventors confirmed the efficiency of the medium formass-production of the present invention by obtaining 10⁸ live bacteriaon the second week of culture. Also, the present inventors confirmedthat the early growth rate was depended on the amount of inoculum butthe gap became smaller as culture progressed (FIG. 4).

Experimental Example 2 Confirmation of the Insecticidal Effect by theDipping Method

The present inventors confirmed the insecticidal effect of Metarhiziumanisopliae HY-2 of the present invention on gold bugs by dipping method.Particularly, shaking cultured Metarhizium anisopliae HY-2 on CDAYmedium for 4 days (26° C., 180 rpm). Measured the density of spores withhemacytometer and adjusted the density to 1×10⁸

using distilled water containing 0.05% tween-80. After dipping 10 larvaeeach from 1^(st) instar, 2^(nd) instar and 3^(rd) instar of Adoretustenuinaculatus in spore suspension for 10 seconds, transferred them toartificial feeds (sawdust:bed soil:water=5:3:2) and raised them at26-28° C. in dark condition. While keeping required humidity for 15days, investigated pathogenesis. At that time, regarded insects on whichhyphae of fungus were generated as dead insects and insects grown tonext instar as live insects. Counted both numbers and presented themwith percentage. All experiments were performed 5 times. Used distilledwater containing 0.05% tween-80 for a control group.

As a result, spore suspension of Metarhizium anisopliae HY-2 showedexcellent insecticidal effect (56-64%) while a control group showed just13-26% insecticidal effect (FIG. 5). In addition, more larvae ofAdoretus tenuinaculatus were confirmed to be infected with Metarhiziumanisopliae HY-2 as time went (FIG. 6).

Experimental Example 3 Confirmation of Insecticidal Effect by theContact Toxicity Test

The present inventors confirmed the insecticidal effect of Metarhiziumanisopliae HY-2 of the present invention on gold bugs by contacttoxicity test. Particularly, shaking cultured Metarhizium anisopliaeHY-2 on CDAY medium for 4 days (26° C., 180 rpm). Put 750 g of solidmedium (wheat bran:rice bran:water=4:2:3) into a polypropylen bag(50×22×10 cm²) having 2 sheets of filter membranes (7×7 cm², effectivearea diameter 3.5 cm) in it for air permeability and then sealed, whichwas autoclaved at 121° C. for 30 minutes. Inoculated 9-10 ml of theculture fluid on the solid medium prepared above and then culturedthereof in a culture room for 3-4 weeks in which temperature wasmaintained at 27° C., the radiation intensity was kept 40 W×2×1.5 andthe relative humidity was kept 40-70%. Measured the density of sporeswith hemacytometer. After crushing the solid medium, adjusted thedensity to 1×10⁸ spores/mt and then distributed thereof over humidifiedartificial feeds (sawdust:bed soil:water=5:3:2). Transferred 10 larvaeeach from 1^(st) instar, 2^(nd) instar and 3^(rd) instar of Adoretustenuinaculatus into the above artificial feeds and raised them at 26-28°C. in dark condition. While keeping required humidity for 15 days,investigated pathogenesis.

At that time, regarded insects on which hyphae of fungus were generatedas dead insects and insects grown to next instar as live insects.Counted those numbers and presented them with percentage. Allexperiments were performed 5 times. Used distilled water containing0.05% tween-80 for a control group.

As a result, Metarhizium anisopliae HY-2 of the present invention showedexcellent insecticidal effect (60-64%) while a control group showed just20-27% insecticidal effect (FIG. 7).

Industrial Applicability

As described hereinbefore, the fungus of Metarhizium genus has excellentinsecticidal effect on larvae of gold bugs, a kind of soil pests harmingagricultural crops, so that it can be effectively used for controllingsoil pests as an environmental-friendly pesticide.

1. A Metarhizium anisopliae fungus designated HY-2 having insecticidalactivity to soil pests, deposited under Gene Bank of Korea ResearchInstitute of Bioscience and Biotechnology under Accession No: KCTC 0156BP.
 2. (canceled)
 3. A medium for mass-production of the fungus ofclaim 1 containing carbon sources, nitrogen sources and inorganicelements.
 4. The medium as set forth in claim 3, wherein the carbonsource is selected from a group consisting of glucose, cornstarch,saccharose, molasses, wheat bran and rice bran, the nitrogen source isselected from a group consisting of yeast extract, soybean flour,corn-immersion, malt extract and peptone, and the inorganic element isselected from a group consisting of potassium chloride, magnesiumsulfonate, ferroic sulfate and sodium nitrate.
 5. A microbialinsecticide for controlling soil pests containing the fungus of claim 1as an effective ingredient.
 6. The microbial insecticide of claim 5,wherein the microbial insecticide is prepared in the forms of wettablepowders, granules or capsules.
 7. The microbial insecticide of claim 6,wherein the wettable powders are prepared by: pulverizing dried solidmedia inoculated with Metarhizium anisopliae HY-2, thereby generating apulverized powder; and adding surfactants and diluents to the pulverizedpowders; and pulverizing there pulverized powder.
 8. The microbialinsecticide as set forth of claim 6, wherein the granules are preparedby the following steps: pulverizing dried solid media inoculated withMetarhizium anisopliae HY-2, thereby generating a pulverized powder;adding surfactants, adjuvant and diluents to the pulverized powder;kneading thereef surfactants, adjuvant, diluents and pulverized powderwith water, thereby generating a dough; and granulating the dough; anddrying the dough.
 9. The microbial insecticide of claim 7, wherein thesurfactant is polycarboxylate, sodium lingo sulfate, sodium dialkylsulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenylether, sodium tripolyphosphate, polyoxyethylene alkyl aryl phosphoricester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkylarylpolymer, polyoxyalkylon alkyl phenyl ether, polyoxyethylene nonylphenyl ether, sodium sulfonate or naphthalene formaldehyde.
 10. Theinsecticide as set forth in claim 6, wherein the granules are preparedwith the following weight ratios: Metarhizium anisopliae HY-2 spores10-60 weight parts, surfactants 2-16 weight parts, nutrients 5-20 weightparts, disintegrators 10-30 weight parts.
 11. The microbial insecticideof claim 10, wherein the granules additionally contained one or morecomponents selected from a group consisting of inactive carriers,preservatives, wetting agents, supply accelerants, attractants,encapsulating agents, binders, emulsifiers, dyes, UV protectors, buffersand fluids.
 12. The microbial insecticide of claim 8, wherein thesurfactant is s polycarboxylate, sodium lingo sulfate, sodium dialkylsulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenylether, sodium tripolyphosphate, polyoxyethylene alkyl aryl phosphoricester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkylarylpolymer, polyoxyalkylon alkyl phenyl ether, polyoxyethylene nonylphenyl ether, sodium sulfonate or naphthalene formaldehyde.